ABSTRACT The pathogenic nature of MeCP2 in the neurodevelopmental disorder Rett syndrome (RTT) has been known for almost 20 years; however, there currently no viable intervention strategies. One contributing factor to the paucity of treatment options is the reliance on male Mecp2-/y knockout mice during preclinical development, despite the fact that RTT almost exclusively affects females, who are mosaic for the x-linked MECP2 allele (Mecp2+/-). Furthermore, RTT patients rarely have mutations that result in loss of MeCP2 protein, rather have missense or truncating mutations that render key functional domains hypomorphic. To address this failing in construct validity, we performed RNA-sequencing on cerebellar and motor cortex samples from 9 RTT patient autopsies to identifying potentially druggable targets which might begin from a place of translational relevance. This approach led us to focus on muscarinic acetylcholine receptors (mAChRs), where 4 of the 5 subtypes had significantly disrupted expression. Of particular interest was the mAChR1 (CHRM1, M1) subtype, which has long been considered a viable therapeutic target for neuropsychiatric diseases. Excitingly, we recently used 44 temporal cortex samples from RTT autopsies to confirm that decreased CHRM1 expression is a robust and highly penetrant aspect of RTT pathophysiology in humans. We then established that administration of an M1 positive allosteric modulator (PAM), VU0453595 (VU595), significantly improves social and respiratory (apnea) phenotypes in Mecp2+/- mice. In Aim 1, we propose to expand these preliminary data by testing the effects of M1 potentiation against a full battery of RTT-like phenotypes in mice, using structurally distinct M1 PAMs, acute and chronic dosing paradigms, and multiple modes of pharmacology. Interestingly, if RTT autopsy expression data is binned by MECP2 mutation, then signature expression patterns are observed, not only for M1 but for virtually all preclinical target genes tested. As changes in gene expression are often the rationale for target selection, and modulation of neurotransmission in contexts where receptor function is normal carries an increased risk for adverse effects, these findings have important implications regarding the need for precision medicine in RTT. In Aim 2, we will use mice carrying common RTT mutations (T158M, R168X, R255X, and R306C) to determine whether expression patterns can be used to predict M1 PAM efficacy. We will couple these experiment with transcript and proteomic analysis of RTT autopsy samples to quantify global gene expression patterns in the medulla of RTT patient sub-populations. Finally, of our existing data set, we consider the effect of VU595 on apneas to be a salient finding, both because respiratory dysfunction is predictive of early lethality in RTT and because apneas represent a highly translatable outcome measure. In Aim 3, we propose to mechanistically dissect the role of M1 in RTT respiratory phenotypes by coupling whole body plethysmography and in vivo cyclic voltammetry experiments in Mecp2 knock-in mice to determine the M1 PAM mechanism of action, as well as define where and how M1 functions in the respiratory circuit across RTT sub-populations.